The long-term goal of this 5-year Competing Continuation Grant Application is to understand the functional regulation of the renal-specific Na+-coupled Cl- cotransporters at a molecular level. The major focus of this application is to determine the molecular mechanisms of regulation of the Na-K-2Cl cotransporter by cyclic AMP [cAMP]. Increasing net NaCl reabsorption in the thick ascending limb of Henle [TAL] by hormones generating cAMP via their respective Gs-coupled receptors is a fundamental mechanism for regulating salt transport in this nephron segment. The effects of these hormones is crucial to the normal functioning of the TAL in reabsorbing 10-15% of filtered NaCl, providing for normal diluting and concentrating power, and regulating divalent mineral excretion. Two major splice variants of the renal-specific Na-K-2CI cotransporter have been recently identified by us and both are expressed in the TAL. The novel hypothesis to be examined in this grant application is that the unique splice variant of the murine Na-K-2CI cotransporter, mBSC1-4, provides the fundamental mechanism for regulating Na-K-2CI cotransporter, mBSC1-9, activity by cAMP-dependent phosphorylation processes. Our working hypothesis is that mBSC1-4 exerts a dominant-negative like effect on mBSC1-9 cotransporter function that is modulated by cAMP-dependent phosphorylation processes. mBSC1-4 is also expressed in the ascending thin limb (aTL) and may serve important transport functions in this nephron segment that is importantly involved in urine dilution and concentration. Using a combination of molecular, biochemical, and physiological approaches, we will: a), characterize the mBSC1-9:mBSC1-4 interactions and the mechanism of cAMP-dependent regulation of Na+ transport [the TAL model]; b) assess mBSC1-4 and mBSCl-9 phosphorylation in the regulation of Na-K-2CI cotransport and mBSC1-4:mBSCl-9 interactions; c), characterize the function and regulation of mBSC1-4 independent of mBSC1-9 [the aTL model]; d), examine the effects of altering water balance or NaCl balance, K+ loading, and alterations in acid-base balance in the regulation of mBSCI-9 and mBSCI-4 in vivo. Results of these studies should begin to define the molecular mechanisms of regulating ion transport by phosphorylation processes in the ascending limb of Henle. This has important relevance to understanding salt and water handling by the kidney in health and disease.